The present invention relates to antigens that mimic the structure of extracellular domains of type III membrane proteins derived from intracellular pathogenic microorganisms, to the conformational antibodies prepared from said antigens, and to the uses thereof for detecting, preventing and treating latent or chronic infections with these microorganisms, and associated pathologies, in particular tumor or autoimmune pathologies.
Certain intracellular microorganisms (viruses, bacteria, fungi, parasites) responsible for latent or chronic infections pose public health problems in many countries; by way of nonlimiting example, mention may be made of viruses such as the hepatitis C virus (HCV), the human immunodeficiency virus (HIV), viruses of the herpesviridae family [Epstein-Barr virus (EBV); cytomegalo virus (CMV); Kaposi's sarcoma herpesvirus (KSHV); herpes simplex virus (HSV); varicella-zoster virus (VZV)] and the hepatitis B virus (HBV), and also intracellular bacteria, in particular Chlamydia trachomatis and Mycobacterium tuberculosis.
These intracellular microorganisms are characterized by their ability to persist in the latent state throughout the life of the host (human or animal) without being eradicated by the immune system in subsequence to the primary infection; this host-virus relationship, which is very complex and poorly elucidated, involves viral or bacterial mechanisms that avoid the immune response, such as genetic variability (HCV, HIV) and the expression of proteins that modulate the immune response [EBNA1 protein (EBV)].
Although infection with these intracellular microorganisms is clinically silent in most individuals, some of them, in particular immunodepressed individuals, develop chronic pathologies, in particular pulmonary (Mycobacterium tuberculosis), genital and ocular (Chlamydia trachomatis) pathologies, tumor pathologies (EBV, HCV, HBV, KSHV) or autoimmune pathologies (EBV). For example, EBV is associated with a large number of tumor pathologies: Burkitt's lymphoma (Central Africa), nasopharyngeal carcinoma (South East Asia), gastric carcinoma, Hodgkin's disease, nasal lymphomas and breast cancer, post-transplantation lymphoproliferative diseases, and AIDS-related lymphomas, and also autoimmune pathologies: rheumatoid arthritis, disseminated lupus erythematosus and Sjögen's syndrome.
The antigens expressed during the chronic phase or latency phase represent target antigens for immunization for preventive or therapeutic purposes, and for the diagnosis of infections with these intracellular microorganisms and of associated pathologies.
For example, latent EBV infection results in the expression of a limited number of viral genes encoding, respectively, for six nucleus proteins (EBNA-1, -2, -3a, -3b and -3c, and LP) and two membrane proteins [LMP1 and LMP2 (LMP2A/LMP2B)]; at least three types of latency (I, II, III) representing at least three distinct expression profiles are associated with distinct tumor pathologies, and a fourth type of latency (IV) representing an expression profile different from the above, could be associated with an asymptomatic state (healthy carriers) (FIG. 1).
It is generally accepted that latent EBV infection is essentially controlled by cellular immunity mediated by a population of CD8+ cytotoxic T lymphocytes (CTLs), specific for EBV latency proteins, essentially for the EBNA proteins, whereas the humoral immunity directed against the EBV latency proteins does not play a protective role in infected individuals. The loss of this control, due either to the decrease in activity of the effectors (CTLs) or to the decrease in target recognition (decrease in class I antigen presentation), would severely compromise the host's ability to control the proliferation of the EBV-infected cells and would be responsible for the development of malignant tumors related to latent EBV infections.
Thus, immunotherapy against EBV-related malignant tumors essentially makes use of the in vitro activation of autologous CTLs, from patients, against latency proteins and of reimplantation thereof, with more or less effectiveness (for a review, see Khanna R. et al., TRENDS in Molecular Medicine, 2001, 7: 270-276). This cell therapy has the drawback of being very expensive and laborious to implement.
Latency antigen detection, which is useful for diagnosing latent EBV infections and associated pathologies, requires a cell permeabilization step before said cells are brought into contact with the antibodies, which is tricky to carry out.
To more effectively combat latent or chronic infections with these intracellular pathogenic microorganisms, and the associated pathologies, there exists a real need for novel antigens for preventive and therapeutic immunization, and also diagnosis.
Among the antigens expressed during the chronic phase or latency phase of the infection, mention may be made of type III membrane proteins, characterized by transmembrane domains that separate short extracellular domains (EDs) and intracellular domains (IDs) and are bordered by larger N- and/or C-terminal intracellular domains; FIG. 2 illustrates the hypothetical structure of type III membrane proteins having 2n membrane domains. By way of nonlimiting example of type III membrane proteins having 2n transmembrane domains, that are expressed during the chronic or latency phase of the infection, mention may be made of the EBV LMP1 and LMP2A proteins (accession number in the Swissprot database, respectively P03230 (SEQ ID NO: 29) and P13285 (SEQ ID NO: 30), with reference to the sequence of the EBV strain B95.8), the KSHV LAMP K15-P and LAMP K15-M proteins (Genbank accession numbers, respectively AAD45297 (SEQ ID NO: 31) and AAD45296 (SEQ ID NO: 32)), the HCV p7, NS2 and NS4B proteins (EMBL accession number AF009606 (SEQ ID NO: 33)), the Chlamydia trachomatis MOMP protein (Major Outer Membrane Protein; NCBI accession number AF352789 (SEQ ID NO: 34)) and the Mycobacterium tuberculosis MmpI 1 to 12 transport proteins.
The absence of antibodies directed against the type III membrane proteins, in individuals suffering latent or chronic infection with these microorganisms, indicates that these proteins are not accessible at the surface of the infected cells or that extracellular domains are not immunogenic (FIG. 2).
The absence of antibodies directed against the extracellular domains, observed when laboratory animals are immunized with recombinant LMP1 and LMP2 proteins, indicates rather that their extracellular domains are not immunogenic (Hennessy et al., K., Proc. Natl. Acad. Sci. USA, 1984, 81: 7207-7211) and with LMP2 (Longnecker et al., J. Virol., 1990., 64: 3219-3226). In fact, all the anti-LMP1 antibodies described recognize more or less well-identified fragments of the intracellular regions of the LMP1 or LMP2A proteins, namely, for LMP1: CS1-4 (pool of 4 monoclonal antibodies, Dako, Glostrup, Denmark), S12 (Dr Elliott Kieff, Harvard Medical School, Boston, Mass.), OT22C and OT22CN (Organon Teknika, Boxtel, The Netherlands). Antibodies directed against extracellular domains of the LMP1 and LMP2 proteins have nevertheless been obtained by immunizing rabbits with synthetic peptides representing the sequence of a single extracellular domain coupled to a carrier protein (European application EP 1229043). However, the antibodies obtained are non-conformational antibodies which do not recognize the natural antigen expressed at the surface of the cells affected by latent EBV infection, but only the denatured antigen or a fragment thereof (linear peptide representing a single extracellular domain). In addition, these antibodies do not exhibit any significant biological activity (complement-dependent cytotoxic activity or ADCC for antibody dependent cellular cytotoxicity).
No antibodies or antigens currently therefore exist that are effective for the treatment (serotherapy or preventive or therapeutic immunization) of latent or chronic infections with the intracellular pathogenic microorganisms as defined above, and of the associated pathologies, in particular EBV infection and the associated tumor pathologies.